1. Field of the Invention
This invention relates to the evaluation of interactions between substances using inverse chromatography methods and apparatus. The term xe2x80x9csubstancexe2x80x9d is used herein, i.e. throughout this specification, to mean a single element or compound, or a mixture of elements and/or compounds. The invention is particularly (but not exclusively) useful in the field of combinatorial science.
2. Introduction to the Invention
In the field of combinatorial science, there is a continuing need for improved methods for characterizing the members of libraries of substances.
The techniques of chromatography are well-known. In one widely used technique, often referred to as liquid solid chromatography, a liquid carrier (the xe2x80x9cmobile phasexe2x80x9d) is passed over a solid (the xe2x80x9cstationary phasexe2x80x9d) in a column. A sample is injected into the mobile phase before the mobile phase enters the column. The sample comprises at least one substance which interacts with the solid. The rate at which the substance migrates through the column depends on its relative affinity for the mobile and stationary phases. The technique can be used to separate two substances with different relative affinities for the phases, to characterize an unknown sample (using one or more known solids), or (in the process sometimes referred to as inverse chromatography) to characterize a known solid (using one or more known samples). Reference may made for example to U.S. Pat. No. 4,869,093 (Gilbert); J. Liquid Chromatography, 3 (3), (1983), 403-417, Schram et al.; Org. Geochem. (1989), 14 (2), 157-164, Antworth et al; Chromatographia 36 (1993), 259-262, Jerebak et al.; Sekitan Kagaku Kaigi Happyo Ronbunshu (1993), 30th, 122-125 (Morino et al.); Conf. Proc. Int. Conf. Coal Sci, 7th (1993, 1, 419-422, Takanohashi et al.; Energy Fuels (1996), 10 (4), 1012-1016, Morino et al.; Energy Fuels (1996), 10 (4), 1017-1021, Kaneko et al.; Anal. Biochem. 242 (1996), 104-111; Brissova et al; and Reactive Polymers 23 (1994), 173-182 Petro et al. Reference may also be made to U.S. Pat. Nos. 6,175,409, 6,406,632, 6,265,226, 6,260,407, 6,294,388 and PCT Publication WO 99/51980. The entire disclosure of each of the patents, publications and patent applications referred to above is incorporated herein by reference for all purposes.
This invention relates to methods and apparatus which are useful for evaluating the interaction between a receptor and a modifying agent in the presence of a liquid carrier, and which make use of novel and inventive variations of the known techniques of liquid solid chromatography. In the terminology often used in chromatography, the invention includes (but is not limited to) methods in which the receptor is the probe and the modifying agent is the sample, and methods in which the modifying agent is the probe and the receptor is the sample.
In a first aspect of the invention, one of the receptor, modifying agent and liquid carrier is a member of a library, preferably either the receptor or modifying agent is a member of a library, and in either case, the method includes evaluating members of the library in a number of test procedures, and using the results of test procedures to select at least one substance for further testing. The substance selected for further testing will generally be a member of the library, but may be a substance having a known relationship to a member of the library.
In other aspects, the invention provides various methods which are useful as test procedures in the first aspect of the invention, but which are also useful in other contexts, for example for evaluation of interaction between substances by inverse chromatography, and for the rapid testing of a sample withdrawn from a continuous process. In yet further aspects, the invention provides apparatus suitable for carrying out the methods of the invention.
The term xe2x80x9cevaluatingxe2x80x9d is used herein in a broad sense to denote any useful assessment of the interaction between the receptor and the modifying agent. For example, in the first aspect of the invention, the term includes any assessment which makes it possible to select at least one substance for further testing. The term xe2x80x9creceptorxe2x80x9d is used herein to denote a substance which can form at least part of a solid phase. Thus, the receptor, alone or in combination with a solid, corresponds generally to the stationary phase in conventional liquid solid chromatography. The term xe2x80x9cliquid carrierxe2x80x9d is used herein to denote any liquid which can be passed continuously over the solid comprising the receptor while the solid is confined in a test chamber. Thus, the liquid carrier corresponds generally to the mobile phase in conventional liquid solid chromatography. The term xe2x80x9cmodifying agentxe2x80x9d is used herein to denote a substance which can form at least part of a liquid sample which can be injected into a stream of the liquid carrier before the stream is passed over the solid in the test chamber. The term xe2x80x9clibraryxe2x80x9d is used herein in a broad sense to denote a plurality of identified substances, typically an associated collection of identified substances. The library is preferably a spatially determinate array of identified substances formed at or in or situated on a common substrate. Preferably, but not necessarily, the members of the library differ from each other in some systematic way, so that the variation of the results of the test procedures can be correlated with those differences. For example, the members can differ from each other in one or more quantified ways. Such differences can be quantified by measurements carried out during preparation of the substances, or by measurements carried out on the substances themselves. The differences can be ascertained before or after members of the library have been selected in accordance with the present invention.
The method of the first aspect of the invention comprises conducting a plurality of test procedures, each of the test procedures comprising the steps of
(a) injecting a liquid test sample comprising the modifying agent into a stream of the liquid carrier, the injection being carried out over a limited time so that a distinct test section of the stream contains the test sample;
(b) passing the stream of liquid carrier containing the test sample over a solid phase in a test chamber, the solid phase comprising the receptor; and
(c) examining the stream of liquid carrier leaving the test chamber to ascertain a variable which reflects the interaction of the modifying agent and the receptor in the presence of the liquid carrier.
Step (c) of the method will often comprise examining a distinct evaluation section of the stream corresponding to the test section in order to ascertain a property of the modifying agent. The term xe2x80x9cto ascertain a property of the modifying agentxe2x80x9d is used herein to include ascertaining a property of the modifying agent itself and/or ascertaining a property of a substance which is produced by the interaction of the modifying agent and the receptor and/or ascertaining a property of part only of the modifying agent.
The results obtained in the steps (c) of the test procedures can optionally be used to select at least one substance for further testing. The further testing can for example involve the interaction between (i) a selected member or members of the library and (ii) the other two of the receptor, modifying agent and liquid carrier used in the test procedures. The further testing can otherwise be unrelated to the original evaluation.
The term xe2x80x9cexamining the stream of liquid carrier leaving the test chamber to ascertain a variable which reflects the interaction of the modifying agent and the receptor in the presence of the liquid carrierxe2x80x9d is used herein in a broad sense to denote any examination which enables the desired evaluation to be made. For example, as further explained below, it may be sufficient to compare the results of the test procedures without quantifying any property of the modifying agent. Preferably (but not necessarily) step (c), alone or in combination with other steps, makes it possible to calculate the proportion of the modifying agent, or a component of the modifying agent, retained in the test chamber.
The objective of the known chromatographic procedures is (i) to achieve at least some separation of the individual components of the sample, followed by collection of at least one separated product, and/or (ii) to achieve a substantial degree of equilibration between the sample and the stationary phase in order to provide detailed characterizing data. The known procedures use, therefore, combinations of stationary and mobile phases, processing times, and columns which achieve these objectives. By contrast, it is not an objective of the present invention to recover a separated product or to achieve equilibrium between the sample and the solid phase. Furthermore, in the first aspect of the invention, the library is preferably examined as quickly as is consistent with selection of a limited number of members for further testing and under dynamic conditions which simulate an ultimate end-use. For example, the objective might be to identify the members of a library having the potential to be additives which produce a desired effect on a fabric being laundered or dry cleaned, or on photographic film being developed, or in a paint being applied to a building material.
Because of these differences in objectives, the test procedures used in the first aspect of the invention often have at least one (i.e. one or more) of the following characteristics which distinguish them from known chromatographic procedures.
(I) The receptor is a substance which has not been used as the stationary phase in liquid solid chromatography. For example, the receptor comprises
(1) a fabric,
(2) at least 50% by weight of particles having an aspect ratio of at least 2 and at least one dimension which is greater than 100 micron,
(3) a substantially pore-free sheet of an organic polymer,
(4) a foodstuff,
(5) a natural or artificial mammalian tissue,
(6) a polysaccharide, a protein or a nucleic acid,
(7) a catalyst,
(8) an elemental metal or metal alloy,
(9) wood,
(10) concrete,
(11) natural or artificial stone,
(12) a semiconductor substance, or
(13) a DNA chip, a molecular recognition chip, or a separation chip.
xe2x80x83The receptor material preferably includes mammalian tissue, either artificial or natural. Other preferred receptor materials are selected from the group consisting of a fabric, a substantially pore-free sheet of an organic polymer, a foodstuff, wood, concrete, natural or artificial stone, and a semiconductor substance. Within this group, receptor materials selected from a fabric, a foodstuff and a semiconductor substance are particularly preferred.
(II) The test chamber is less efficient than columns used in liquid solid chromatography. For example, the efficiency of the test chamber is less than 80, preferably less than 50, particularly less than 10, theoretical plates.
(III) The test procedures are carried out rapidly and under dynamic conditions. These dynamic conditions are not intended to promote separation of the modifying agent into component parts or to ensure that all of the modifying agent passes through the test chamber. Furthermore, the extent of the interaction between the receptor and the modifying agent can vary widely in the different test procedures. As a result, one or more of the following conditions are often fulfilled.
(1) In each of the test procedures, the test sample comprises an initial quantity of the modifying agent, and in at least one of the test procedures at least 1%, often at least 10%, e.g. 20-70% or 30-50%, of the initial quantity of the modifying agent is retained in the test chamber. Substantially all of the modifying agent retained until the end of a test procedure remains retained on the solid phase during subsequent test procedures, and thus can be regarded as being irreversibly retained on the solid phase under the test conditions.
(2) In each of the test procedures, the test sample comprises an initial quantity of the modifying agent, and the difference between (i) the lowest percentage of the initial quantity of modifying agent retained in the test chamber in any of the test procedures and (ii) the highest percentage of the initial quantity of modifying agent retained in the test chamber in any of the test procedures, is at least 10, for example 10-70, often at least 25, for example 30-60.
(3) In at least one, preferably in each, of the test procedures, the receptor is not saturated by the modifying agent. The extent to which saturation is being approached can be assessed by repeating one of the test procedures. Preferably, in such a repeated test procedure, the same proportion of the modifying agent is retained by the receptor, indicating that the receptor is far from saturation. If a lesser proportion is retained, this indicates that the receptor is approaching saturation; and when none of the modifying agent is retained, the receptor is saturated.
(4) In at least one, preferably in each, of the test procedures, a plot of time against concentration of the modifying agent in the stream of liquid carrier leaving the test chamber has only a single peak, with the slope of the plot being positive at all points on one side of the peak and negative at all points on the other side of the peak.
(5) In at least one, preferably in each, of the test procedures, the stream of liquid leaving the test chamber is passed through a detector, and the time taken for the evaluation section to pass through the detector is less than five times the time taken for the test section containing the modifying agent to pass through the entrance to the test chamber.
(IV) The desired evaluation is carried out merely by ascertaining the proportion of the modifying agent which is retained in the test chamber (i.e. it is unnecessary to know, in absolute terms, the quantity of modifying agent initially present in the sample). This makes it possible to carry out the desired evaluation by comparing (a) the results of passing the sample-carrying liquid stream through the test chamber and (b) the results of a reference procedure in which a similar sample-carrying liquid stream is passed through a reference chamber which (i) is free of any substance which interacts with the modifying agent, or (ii) contains a known solid substance. In some cases, satisfactory results can be obtained by comparing the results of each of the test procedures with a single reference procedure or with two or more reference procedures which are carried out at appropriate intervals. However, this requires a uniformity between the different procedures which is not always easy to attain. It is preferred, therefore, that each of the test procedures should itself incorporate the reference procedure. In this case, each of the test procedures further comprises
(d) injecting a liquid reference sample into a second stream of the liquid carrier, the composition of the second sample being substantially identical to the composition of the test sample, and the injection being carried out over a limited time so that only a distinct reference section of the stream contains the reference sample;
(e) passing the second stream of liquid carrier containing the reference sample through a reference chamber which is free of any substance which interacts with the modifying agent;
(f) examining the stream of liquid carrier leaving the reference chamber to ascertain a property of the modifying agent remaining in the stream; and
(g) comparing the results obtained in steps (c) and (f) to evaluate the interaction between the receptor and the modifying agent.
In the aforementioned protocol, the second stream into which the reference sample is injected can be the same stream into which the test sample was injected (or alternatively, can be a separate and independent stream (i.e., a separate line)). Preferably, therefore, the first liquid stream containing the test section and second liquid stream containing the reference section are obtained by
(i) injecting into the stream of liquid carrier a liquid unit whose composition is the same as the composition of the test and evaluation samples and whose size is equal to the sum of the sizes of the test and evaluation samples, the injection being carried out over a limited time so that only a distinct section of the liquid stream contains the liquid unit, and
(ii) splitting the liquid stream containing the liquid unit into a first sub-stream which passes through the test chamber and includes the test section and a second sub-stream which passes through the reference chamber and includes the reference section.
In the methods of the first aspect of the invention, the test procedures can be carried out in series or in parallel or both.
In some embodiments, it is particularly preferably to use parallel (simultaneous) procedures, especially with respect to applications in combinatorial materials science, in which either the library or receptors are members of a combinatorial library comprising at least four members, such that members are evaluated for interaction between a receptor member of the combinatorial library and a modifying agent, or alternatively for interaction between a modifying agent member of a combinatorial library and a receptor, in each case in the presence of a liquid carrier. Preferably each of the at least four members of the combinatorial library comprise a different non-biological polymer. The method includes conducting at least four test procedures in parallel under a common set of test conditions, with the receptor, modifying agent and liquid carrier being the same in each of the test procedures except that in each test procedure a different member of the combinatorial library is used.
The parallel testing/evaluation can include multiple flow channels with simultaneous contacting of test samples (comprising a modifying agent) and solid phases (comprising a receptor) as compared between different channels. The injection into such parallel-configured carrier streams, each having its own dedicated test chamber, can be accomplished in a sequential manner, as described for example in co-owned U.S. Pat. No. 6,296,771. Alternatively, the injection into such parallel-configured carrier streams can also be accomplished in parallel, using parallel injection systems such as are disclosed in co-owned, co-pending U.S. applications, Ser. No. 09/641,442 filed Aug. 2, 2002 by Freitag et al. and Ser. No. 10/092,035 filed Mar. 6, 2002 by Bergh et al. In either case, the detection (examining step) is preferably done in parallel, but can also be sequential. In a preferred protocol, the at least four parallel test procedures can comprise:
(a) simultaneously injecting at least four liquid test samples comprising an initial quantity of the modifying agent into at least four separate and distinct streams of the liquid carrier, respectively, the injections being carried out over a limited time so that distinct test sections of the at least four streams contain the test sample,
(b) simultaneously passing the at least four streams of liquid carrier containing the test samples over a solid phase in at least four separate and distinct test chambers, respectively, the solid phase comprising the receptor,
(c) retaining at least 10% of the initial quantity of the modifying agents on the solid phase in each of the at least four test chambers, the retained modifying agents being irreversibly retained under the test conditions of the procedures, and
(d) simultaneously examining the at least four streams of liquid carrier leaving the test chamber to ascertain, for each of the at least four streams, a variable which reflects the interaction of the modifying agent and the receptor in the presence of the liquid carrier for that respective stream, and
(e) comparing the interaction of the modifying agent and the receptor for each of the at least four streams to determine a relative ranking of the members of the combinatorial library with respect to such interaction.
It is specifically contemplated that such parallel methods, and in particular the above-detailed preferred parallel methods for evaluation of combinatorial libraries can be used in connection with each and every other embodiment disclosed herein (e.g., evaluating interaction between a modifying agent and a receptor comprising natural or artificial mammalian tissue, or other types of materials (as listed above), evaluation protocols involving a reference chamber, etc.)xe2x80x94such as test procedures (I) to (IV) as outlined above.
Individual test procedures having one or more of characteristics (I) to (IV) set out above are in themselves novel and inventive, and form part of the present invention. Thus, in second to fifth aspects, the present invention provides methods which are suitable for evaluating the interaction between a receptor and a modifying agent in the presence of a liquid carrier and which respectively have one of characteristics (I) to (IV) set out above.
In a sixth aspect, the invention provides apparatus suitable for carrying out the method of the first aspect of the invention wherein the modifying agent is one of the library and each of the test procedures has characteristic (I) above, the apparatus comprising
(A) a test chamber for the solid phase comprising a receptor as defined in characteristic (I) above;
(B) a reservoir for the liquid carrier;
(C) a pump for continuously extracting a stream of the liquid carrier from the reservoir and passing the stream through the test chamber;
(D) an autodilution and sampling robot for sequentially injecting into the stream of liquid carrier, before the stream passes through the chamber, a plurality of liquid test samples, each sample containing an initial quantity of one of the library of modifying agents and each sample being injected over a limited time so that only a distinct test section of the liquid stream contains the sample; and
(E) a detector for examining the stream of liquid carrier leaving the test chamber without removing anything from the stream and for determining for each sample the proportion of the initial quantity of the modifying agent remaining therein.
In a seventh aspect, the invention provides apparatus suitable for carrying out the method of the first aspect of the invention wherein the method has characteristic (II) above, the apparatus comprising a test chamber which contains the solid phase comprising the receptor and which has an efficiency of less than 80, preferably less than 50, especially less than 10, theoretical plates; and a reservoir, pump, autodilution and sampling robot and detector as in the apparatus of the seventh aspect of the invention.
In an eighth aspect, the invention provides apparatus which is suitable for carrying out the method of the first aspect of the invention wherein the method has characteristic (IV) as defined above, and which comprises
(A) a test chamber containing the solid phase comprising the receptor;
(B) a reference chamber which is free of any substance which interacts with the modifying agent;
(C) a reservoir for the liquid carrier;
(D) a main passageway from the reservoir;
(E) a first branch passageway which is connected to the main passageway has a junction and which leads from the main passageway to the test chamber;
(F) a second branch passageway which leaves from the test chamber;
(G) a third branch passageway which is connected to the main passageway at the junction and which leads from the main passageway to the reference chamber;
(H) a fourth branch passageway which leaves from the reference chamber;
(I) an exit passageway which combines the second and fourth branch passageways;
(J) a pump for extracting a stream of the liquid carrier from the reservoir and passing it through the main passageway;
(K) an injector for injecting into the stream of liquid carrier, before the stream passes from the main passageway to the first and third branch passageways, a liquid unit containing an initial quantity of the modifying agent, the unit being injected over a limited time so that only a distinct section of the liquid stream contains the unit, and the sample being divided into a first sub-stream which contains a first sample of liquid unit and which passes through the test chamber and a second sub-stream which contains a second sample of the liquid unit and which passes through the reference chamber; and
(L) a detector for examining the stream of liquid carrier passing out of the exit passageway and for ascertaining the proportion of the modifying agent remaining therein.